Anti-GFP/BrdU In Situ Immunostaining

Posted on August 1st, 2016 by UNM CC

<R. Gu 4/6/2005

Materials

24 well dishes
Millicell 0.4 µm inserts (Millipore PICM01250) - coated with fibronectin
Culture medium
PBS
Fixative: 4% paraformaldehyde in PBS (pH 7.6)
Rabbit anti-GFP (Molecular Probes, A-11122)
FITC-goat anti-rabbit IgG (Jackson ImmunoResearch, 111-096-006)
Biotinylated anti-BrdU (AbCam, ab2284-125)
Streptavidin-Alexa Flour 555 (Invitrogen/Molecular Probes, S21381)
Goat serum
Horse serum
BSA
2N HCl
DAPI Fluorescent mounting medium (Vectashield, Vector Labs H-1200)
Glass microscope slides and cover slips

Procedure

  1. Coat Millicell inserts with fibronectin as described by Millipore.
  2. Cultivate tissue at 37ºC on fibronectin coated well inserts in a 24-well dish.
  3. Remove well inserts and cut out mesh insert with a scalpel, forceps and dissecting microscope
  4. Place inserts in a 2.0 mL Eppendorf tube with 1 mL of blocking agent (10% goat serum, 1% BSA in PBS). Gently flick tube to mix.
  5. Remove solution and wash 3x by adding 1 mL PBS, 3' each.
  6. Add 0.3 mL rabbit anti-GFP (1:200 v/v, plus 1% goat serum in PBS) and incubate 1 hr. at room temp. Wash 3x in PBS.
  7. Post-fix in 1 mL 4% paraformaldehyde x15', room temp. Wash 3x in PBS.
  8. Denature DNA with 1 mL of 2 N HCl x30'. Wash 3x in PBS.
  9. Block in 1 mL 10% Horse serum, x30' at room temp. Wash 1x in PBS.
  10. Add 0.3 mL Biotinylated anti-BrdU (1:300, with 1% horse serum in PBS). Incubate at 4ºC o.n. or 2 hr. at room temp. Wash 3x in PBS.
  11. Dilute 1 µL fresh streptavidin-Alexa Flour 555 in 1 mL PBS (1:1,000). Spin the dilute antibody at high speed for 30' to remove particulates. Add supernatant to specimen and incubate 30' at room temp.
  12. Remove antibody and add 1 mL PBS. O.K. to store at 4ºC.
  13. Use a scalpel to remove membrane from well inserts. Spot 25-50 µL of DAPI mounting medium onto a glass slide. Place 1-2 membranes on the DAPI medium. Use a dissecting microscope to be sure to place the tissue side up. Cover with a 24-40 mm glass slip. Paint the edges with clear nail polish. View with immunoflourescence. Store in the dark at 4ºC.

Tags: Fero-Histology

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